Biochemical analysis and crystallisation of Fc gamma RIIa, the low affinity receptor for IgG

Fc gamma RIIa is one of a family of specific cell surface receptors for imm unoglobulin. Fc gamma RIIa, which binds immune complexes of certain IgG iso types, plays important roles in immune homeostasis. However, the precise ch aracteristics of IgG binding and three-dimensional structure of Fc gamma RI Ia have not been reported. This study describes the affinity of the Fc gamm a RIIa:IgG interaction as well as biochemical characterisation of recombina nt Fc gamma RIIa that has been used to generate high quality crystals. Equi librium binding analysis of the Fc gamma RII:IgG interaction found, IgG3 bi nds with an affinity of K-D = 0.6 mu M, as expected. Unlike other Fc gamma R, IgG4 also bound to Fc gamma RIIa, K-D = 3 mu M, clearly establishing Fc gamma RIIa as an IgG4 receptor. Biochemical analysis of mammalian and insec t cell derived Fc gamma RIIa established the genuine N-terminus with Q bein g the first amino acid in the sequence Q, A, A, A, P... extending the N-ter minus further than previously thought. Furthermore, both potential N-linked glycosylation sites are occupied. Electrospray ionisation mass spectrometr y (ESMS) indicate that the N-glycans of baculovirus derived Fc gamma RIIa a re core mannose oligosaccharide side chains. Finally, we describe the first crystallisation of diffraction quality crystals of soluble Fc gamma RIIa. Orthorhombic crystals diffract. X-rays beyond 2.1 Angstrom resolution in th e space group P2(1)2(1)2 with cell dimensions a = 78.8 Angstrom, b = 100.5 Angstrom, c = 27.8 Angstrom. This marks a significant advance towards under standing the three-dimensional structure of Fc gamma RIIa and related FcR p roteins that share high amino acid identity with Fc gamma RIIa. (C) 1999 El sevier Science B.V. All rights reserved.